Thymidylate synthase is an essential enzyme involved in nucleotide metabolism. It converts uridylate to thymidylate and provides the sole source of thymidylate for DNA biosynthesis. Since thymidylate is required for DNA biosynthesis and repair, thymidylate synthase represents an attractive target for anticancer agents. Inhibition of thymidylate synthase results in a thymineless state, which is cytotoxic to actively dividing cells. The increased growth rates of cancer cells makes them more sensitive to thymidylate synthase inhibitors than normal cells. Drugs targeting thymidylate synthase are useful against colorectal cancer, gastrointestinal, breast, head and neck, and ovarian cancers (Brandt, D. S., et al., Oncol. Res. 1997, 9, 403-410). The major class of drugs used to target thymidylate synthase is the fluorinated pyrimidines. Fluorinated pyrimidines, including 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FUdR), compete with uridylate for binding to thymidylate synthase. In cells, 5-fluorouracil is converted to FdUMP. FdUMP takes the place of thymidylate and forms a tight binding complex with thymidylate synthase and 5,10-methylene tetrahydrofolate. 5-fluorouracil is the drug of choice for colorectal cancer. In general, response rates with 5-fluorouracil are approximately 10-15% (Brandt, D. S., et al., Oncol. Res. 1997, 9, 403-410).
The major problem with these types of anticancer drugs is the frequent development of drug resistance. A common mechanism by which this occurs is an increased synthesis of thymidylate synthase. Johnston, P. G., et al. (Cancer Res. 1995, 55, 1407-1412) demonstrated a correlation between increased thymidylate synthase gene and protein expression and a decreased responsiveness to 5-fluorouracil. Chu, E., et al. (Proc. Natl. Acad. Sci. USA 1991, 88, 8977-8981) demonstrated that translation of thymidylate synthase mRNA is controlled by its protein product in a negative autoregulatory manner. The binding of the thymidylate synthase protein to its mRNA prevents the translation of the mRNA. Thus drugs that bind to thymidylate synthase can result in increased expression of thymidylate synthase. Thymidylate synthase has also been associated with multidrug resistance. Chu, E., et al. (Mol. Pharmacol. 1991, 39, 136-143) show that cancer cell lines grown to be adriamycin-resistant developed increased expression of thymidylate synthase and showed resistance to 5-fluorouracil. Other inhibitors of thymidylate synthase have been developed in the hope that drug resistance will be less common. Several are in phase I clinical trials, with one (Tomudex, also raltitrexed; ICI D1694; N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2 -theonyl-L-glutamic acid)) in phase III clinical trials. However, the autoregulatory mechanism of thymidylate synthase suggests that drug resistance will not be easily overcome. In fact, resistance to Tomudex has already been seen (Johnston, P. G., et al., J. Natl. Cancer Inst. 1995, 87, 1558-1559).
Combinational therapy is a rational approach with thymidylate synthase inhibitors. One such relevant combination is 5-fluorouracil and leucovorin. Leucovorin is a precursor to 5,10-methylene tetrahydrofolate, and thus also binds to thymidylate synthase. Van der Wilt, C. L., et al. (Cancer Research 1992, 52, 4922-4928) found that in mouse cancer cell lines, leucovorin and 5-fluorouracil combination could prevent increases in thymidylate synthase expression. In a clinical trial, the combination increased the response rate to 20-30% (Rustum, Y. M., et al., J. Clin. Oncol. 1997, 15, 389-400).
The most common approach to targeting thymidylate synthase is the use of chemical compounds that bind to the enzyme. Although there are numerous compounds in clinical trials in an effort to achieve improved response rates compared to 5-FU or Tomudex, it is likely that resistance to these drugs will occur. Monoclonal antibodies against human thymidylate synthase are disclosed in U.S. patent application Ser. No. 07/690,841, but monoclonal antibodies typically generate an immune response against the antibody itself and thus have drawbacks for clinical use.
Oligonucleotides represent a novel approach that target the mRNA encoding thymidylate synthase, rather than the enzyme itself. Such an approach should circumvent the autoregulation of thymidylate synthase protein levels. Kobayashi, H., et al. (Jpn. J. Cancer Res. 1995, 86, 1014-1018) designed a ribozyme targeted to a triple tandem CUC repeat in the 5' UTR of the thymidylate synthase gene, cloned the ribozyme into a vector, and transfected the construct into a B cell lymphoblastoid cell line. They found that cell lines transfected with the ribozyme became sensitive to thymidylate synthase inhibitors. In addition, mRNA expression was reduced compared to control cells.
The use of antisense compounds represents a novel approach distinct from the use of ribozymes. An antisense oligonucleotide has been disclosed that targets the thymidylate synthase portion of the bifunctional dihydrofolate reductase-thymidylate synthase of Plasmodium falciparum, a causative agent of malaria [Sartorius, C., et al., Nucl. Acids. Res. 1991, 19, 1613-1618). Additional chemically-modified antisense oligonucleotides, including phosphorothioate, phosphodiester-phosphorothioate hybrids, and 2'-O-methyl-2'-deoxy chimeras, to the P. falciparum thymidylate synthase are disclosed by Barker, Jr., R. H., et al. (Exper. Parasitology 1998, 88, 51-59). Ju, J-F., et al. (Proc. Amer. Assoc. Cancer Res. 1997, 38, 478) showed that an oligonucleotide targeted to the translation start site of thymidylate synthase mRNA increased the cellular level of thymidylate synthase protein following an initial inhibition of translation. However, oligonucleotide sequences were not disclosed.
There remains a need for improved compositions and methods for modulating human thymidylate synthase gene expression.